Phosphorylation at Serines 216 and 221 Is Important for <i>Drosophila HeT-A</i> Gag Protein Stability

<div><p>Telomeres from <i>Drosophila</i> appear to be very different from those of other organisms – in size and the mechanism of their maintenance. In the absence of the enzyme telomerase, <i>Drosophila</i> telomeres are maintained by retrotransposition of three elements, <i>HeT-A</i>, <i>TART</i>, and <i>TAHRE</i>, but details of their transposition mechanisms are not known. Here we characterized some biochemical characteristics of the <i>HeT-A</i> Gag protein encoded by the <i>HeT-A</i> element to understand this mechanism. The <i>HeT-A</i> Gag protein when overexpressed in S2 cells was localized to the nucleus but was resistant to high salt, detergents and nuclease extraction treatments. Analysis of the <i>HeT-A</i> Gag protein by tandem mass spectrophotometry revealed that serines 216 and 221 are phosphorylated. Substituting these serines with alanine or aspartic acid by site-directed mutagenesis did not result in any changes in <i>HeT-A</i> Gag translocation across the nucleus, suggesting that phosphorylation of these sites is not associated with <i>HeT-A</i> Gag translocation, but time course experiments showed that these phosphorylation sites are important for Gag-protein stability.</p></div

Confirmation of stable cell lines expressing <i>HeT-A</i> Gag proteins.

<p>(A) The stable cell lines were confirmed by isolating genomic DNA from cells and using a vector specific primer (PMK33-1) and an insert-specific primer (9D4 R4) by PCR. 10 ul of PCR product was run on 1% agarose gel and stained with ethidium bromide and photographed (Lane 1 =  Pos, Lane 2 = S2 cells only, Lane 3 =  Vector alone, Lane 4 =  wild type <i>HeT-A</i> Gag, Lane 5 = M1 Gag, Lane 6 = M2 Gag, Lane 7 = M3 Gag). (B) Wild type and mutant <i>HeT-A</i> Gag stable cell lines expressed the same size Gag proteins as confirmed by immunoblot. Expression was only seen when cells were induced with CuSO₄ (lane 1 = S2 cell, Lane 2 =  Vector alone, Lane 3 =  wild type Gag, Lane 4 = M1 Gag, Lane 5 = M2 Gag, Lane 6 = M3 Gag. (C) In wild type and mutant stable cell lines Gag protein was localized in the nuclear fraction as confirmed by immunoblot.</p

Making of a <i>HeT-A</i> Gag-FLAG stable cell line.

<p>(A) Line diagram of the PMK33 vector showing important genetic markers with <i>HeT-A</i> Gag ORF1 cloned at the SmaI restriction site and a 3x FLAG tag at the C-terminus. (B) Expression of <i>HeT-A</i> Gag-FLAG in S2 cell lines as analyzed by PCR and immunoblot. GAPDH and actin serve as internal controls for PCR and immunoblots respectively. (C) Stability of expressed <i>HeT-A</i> Gag-FLAG protein in S2 cells. Cells were seeded in a T-25 flask and induced with 500 µM CuSO4. 1.0 ml cells were collected each day for a total of 6 days and analyzed by immunoblotting. (D) <i>HeT-A</i> Gag-FLAG is targeted to the nucleus. Stably transfected S2 cells were induced with CuSO₄ and 48 hr post-induction cytoplasmic and nuclear fractions were analyzed by immunoblot. (E) <i>HeT-A</i> Gag-FLAG is only detected in stably transfected S2 cells and not in S2 cells. Cytoplasmic, nuclear, and nuclei fractions from both S2 and stably transfected S2 cells were analyzed by immunoblot (1 =  cytosolic, 2 =  nuclear, and 3 =  nuclei).</p

Tandem MS analysis of <i>HeT-A</i> Gag protein by mass spectrophometry.

<p>(A) MS/MS of ion m/z shows an extensive y-ion series and the presence of phosphorylation at serines 216 or 221, as indicated by the arrow. (B) Line diagram of different forms of <i>HeT-A</i> Gags (M1, M2, and M3) that were generated by site directed mutagenesis. Primer sets and template used for site directed mutagenesis are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075381#pone-0075381-t002" target="_blank">Table 2</a>.</p

Time course of <i>HeT-A</i> Gag protein expression and proteasome treatment.

<p>(A) For the time course study, wild type, M1, M2, and M3 cells were seeded in T-25 flaks. At 80% confluence, cells were treated with 500 µM CuSO₄, and equal numbers of cells were removed every 24 hrs for 5 days. Collected cells were lysed in SDS-PAGE buffer, subjected to immunoblotting, and the protein was detected by anti-FLAG antibody. Each membrane was stripped and probed for actin as a loading control. (B) For the proteasome treatment study, cells were seeded and induced as above. 72 hrs post CuSO₄ inductions wild type, M2, and M3 cells were treated with 10 µM of proteasome inhibitor (MG132). Equal numbers of cells were harvested for various time points and analyzed by immunoblot.</p

Primers for PCR, sequencing, and site-directed mutagenesis.

<p>Primers for PCR, sequencing, and site-directed mutagenesis.</p

Mutagenesis sites, primers and templates for site-directed mutagenesis.

<p>Mutagenesis sites, primers and templates for site-directed mutagenesis.</p

Family composition according to mean (median/Interquartile range) number of children per age band.

<p>Family composition according to mean (median/Interquartile range) number of children per age band.</p

Incidence of IHD among cases and controls (COHORT B)^*.

<p>¹incorporates IHD, CHD, angina and MI</p><p>*Analysis excludes cases of IHD diagnosed prior to IBD diagnosis.</p><p>Incidence of IHD among cases and controls (COHORT B)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139745#t004fn002" target="_blank">*</a>}.</p

Incidence Rate Ratios (IRR) comparing whole group analysis with sub-set^*.

<p>*Analysis excludes cases of IHD diagnosed prior to IBD diagnosis.</p><p>Incidence Rate Ratios (IRR) comparing whole group analysis with sub-set^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139745#t006fn001" target="_blank">*</a>}.</p